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Arbuscular mycorrhizal fungi (AMF) live in symbiosis with plant roots, facilitating mineral nutrient transfer from soil to hosts through large networks of extraradical hyphae. Limited data are available on the fungal structures (appressoria) connecting soil- to root-based mycelium, in relation to plant nutrition. Two in vivo systems were set up using three AMF, Funneliformis mosseae, Funneliformis coronatus and Rhizoglomus irregulare, grown in symbiosis with Cichorium intybus. The assessment of plant P content, number of appressoria, diameter of their subtending hyphae and length of colonized roots allowed calculation of the total cross-section area of appressorium-subtending hyphae, which differed among the three AMF and was correlated with plant P contents and with extraradical mycelium density. A conservative evaluation of P fluxes from soil- to plant-based hyphae occurring through appressoria gave values ranging from 1.7 to 4.2 × 10 -8  mol cm -2  s -1 (moles per total cross-section area of the appressorium subtending hyphae per time elapsed), depending on AMF identity. This work suggests that, beyond intraradical colonization and extraradical mycelium extent, connections between extraradical and intraradical fungal mycelium through appressoria are important for mycorrhizal plant nutrition, as appressorium structural traits and density can be related to P transfer mediated by AMF.

Arbuscular mycorrhizal fungi (AMF) are widespread, important plant symbionts. They absorb and translocate mineral nutrients from the soil to host plants through an extensive extraradical mycelium, consisting of indefinitely large networks of nonseptate, multinucleated hyphae which may be interconnected by hyphal fusions (anastomoses). This work investigated whether different lineages of the same isolate may lose the ability to establish successful anastomoses, becoming vegetatively incompatible, when grown separately. The occurrence of hyphal incompatibility among five lineages of Funneliformis mosseae, originated from the same ancestor isolate and grown in vivo for more than 20 years in different European locations, was assessed by systematic detection of anastomosis frequency and cytological studies. Anastomosis frequencies ranged from 60 to 80% within the same lineage and from 17 to 44% among different lineages. The consistent detection of protoplasm continuity and nuclei in perfect fusions showed active protoplasm flow both within and between lineages. In pairings between different lineages, post-fusion incompatible reactions occurred in 6-48% of hyphal contacts and pre-fusion incompatibility in 2-17%. Molecular fingerprinting profiles showed genetic divergence among lineages, with overall Jaccard similarity indices ranging from 0.85 to 0.95. Here, phenotypic divergence among the five F. mosseae lineages was demonstrated by the reduction of their ability to form anastomosis and the detection of high levels of vegetative incompatibility. Our data suggest that potential genetic divergence may occur in AMF over only 20 years and represent the basis for detailed studies on the relationship between genes regulating anastomosis formation and hyphal compatibility in AMF.

Arbuscular mycorrhizal fungi (AMF) establish beneficial mutualistic symbioses with land plants, receiving carbon in exchange for mineral nutrients absorbed by the extraradical mycelium (ERM). With the aim of obtaining in vivo produced ERM for gene expression analyses, a whole-plant bi-dimensional experimental system was devised and tested with three host plants and three fungal symbionts. In such a system, Funneliformis mosseae in symbiosis with Cichorium intybus var. foliosum, Lactuca sativa, and Medicago sativa produced ERM whose lengths ranged from 9.8 ± 0.8 to 20.8 ± 1.2 m per plant. Since ERM produced in symbiosis with C. intybus showed the highest values for the different structural parameters assessed, this host was used to test the whole-plant system with F. mosseae, Rhizoglomus irregulare, and Funneliformis coronatus. The whole-plant system yielded 1-7 mg of ERM fresh biomass per plant per harvest, and continued producing new ERM for 6 months. Variable amounts of high-quality and intact total RNA, ranging from 15 to 65 μg RNA/mg ERM fresh weight, were extracted from the ERM of the three AMF isolates. Ammonium transporter gene expression was successfully determined in the cDNAs obtained from ERM of the three fungal symbionts by RT-qPCR using gene-specific primers designed on available (R. irregulare) and new (F. mosseae and F. coronatus) ammonium transporter gene sequences. The whole-plant experimental system represents a useful research tool for large production and easy collection of ERM for morphological, physiological, and biochemical analyses, suitable for a wide variety of AMF species, for a virtually limitless range of host plants and for studies involving diverse symbiotic interactions.

Arbuscular mycorrhizal fungi (AMF) live in symbiosis with most plant species and produce underground extraradical hyphal networks functional in the uptake and translocation of mineral nutrients from the soil to host plants. This work investigated whether fungal genotype can affect patterns of interconnections and structural traits of extraradical mycelium (ERM), by comparing three Glomeraceae species growing in symbiosis with five plant hosts. An isolate of Funneliformis coronatus consistently showed low ability to form interconnected ERM and self-incompatibility that represented up to 21% of hyphal contacts. The frequency of post-fusion self-incompatible interactions, never detected before in AMF extraradical networks, was 8.9%. In F. coronatus ERM, the percentage of hyphal contacts leading to perfect hyphal fusions was 1.2-7.7, while it ranged from 25.8-48 to 35.6-53.6 in Rhizophagus intraradices and Funneliformis mosseae, respectively. Low interconnectedness of F. coronatus ERM resulted also from a very high number of non-interacting contacts (83.2%). Such findings show that AMF genotypes in Glomeraceae can differ significantly in anastomosis behaviour and that ERM interconnectedness is modulated by the fungal symbiont, as F. coronatus consistently formed poorly interconnected networks when growing in symbiosis with five different host plants and in the asymbiotic stage. Structural traits, such as extent, density and hyphal self-compatibility/incompatibility, may represent key factors for the differential performance of AMF, by affecting fungal absorbing surface and foraging ability and thus nutrient flow from soil to host roots.